Method for extracting lutein from green plant materials

ABSTRACT

The present invention provides a method for extracting carotenoids from green plant materials using supercritical fluid extraction. A first and second supercritical fluid extraction is performed on the green plant composition at two different pressures to obtain two extracts. The first extract includes substantial amounts of β-carotene. The second extract is substantially free of β-carotene, and includes substantial amounts of lutein.

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of natural productextraction. More particularly, it concerns the use of supercriticalfluid for the extraction of carotenoids from green plant materials.

Carotenoids are highly colored naturally occurring compounds, which arewidely distributed in nature. Carotenoids may be classified ashydrocarbon carotenes or xanthophylls, which are oxygenated derivativesof carotenes. Representative examples of carotenes include β-carotene,alpha-carotene, and lycopene. Examples of xanthophylls include lutein,astaxanthin, canthaxanthin, zeaxanthin, and capsorubin. Carotenoids havebeen shown to have anti-oxidant properties and have been studied for theprevention of cancer and other human diseases.

Carotenoids are naturally present in edible leaves, flowers, and fruits,and are readily obtained from flowers (i.e. marigold), berries, and roottissue (i.e. carrots). Hydrocarbon carotenes, such as β-carotene andlycopene, are typically present in an uncombined free form, which isentrapped within chloroplast bodies within plant cells. Xanthophylls,such as lutein, are abundant in a number of yellow or orange fruits andvegetables such as peaches, mango, papaya, prunes, acorn squash, andoranges. Some Xanthophylls are present in plant flowers, such asmarigolds, as long chain fatty esters, typically diesters, of acids suchas palmitic and myristic acids. Generally, the free forms of carotenoidsare present in the chlorplasts of green plants such as alfalfa, spinach,kale and leafy green plant materials. The free form of the carotinoidsprovides better adsorption when consumed in foods or as a supplement.

Lutein is a xanthophyll found in high concentrations in the macula ofthe eye and in the central part of the retina. It serves important rolesin vision to help filter ultraviolet wavelengths of light to preventdamage to the eye lens and macula. Lutein's antioxidant properties arebelieved to help protect the macula, which is rich in polyunsaturatedfats, from light-induced free radicals. Lutein can not be produced bythe body, and consequently, must be ingested. Thus, lutein has becomeincreasingly used in nutritional supplements for the prevention and/ortreatment of vision losses due to macular degeneration, cataracts andretinitis pigmentosa.

Lutein has been shown to have significant potential in the prevention ofage-related macular degeneration (AMD), the leading cause ofirreversible blindness among Americans age 65 and older. Lutein helpsbuild macular pigment density, a critical factor in the health of themacula and the retina. It has been found that high intake of lutein-richgreen plants (spinach and kale) reduce the rate of AMD by 40% whereasBeta-carotene, vitamin A, zinc, and vitamin E were not seen to have aneffect (Seddon et al. 1994). It has been shown that the accumulation oflutein in the macular pigment is dependent upon dietary intake and thatthe density of the macular pigment is related to the preservation ofvisual sensitivity and protection from AMD (Pratt, 1999, Richer, 2001).Other vision loss problems, such as cataracts and retinitis pigmentosamay also be stopped or reduced with a high intake of lutein.

The most common source of extracted lutein is from marigold flowerpetals, which contain one of the highest levels of lutein known and havea low concentration of other carotenoids. Methods of the purification oflutein-fatty acid esters from marigold flower petals are reported inU.S. Pat. Nos. 4,048,203, 5,382,714 and 5,648,564, in which dried groundmarigold flower petals are extracted with a hydrocarbon solvent. In U.S.Pat. No. 5,648,564, extraction is performed 8-10 times with a 60-minutesoak in hexane solvent for the extraction of the carotenoid from themarigold, and uses 320-400 L hexane for each 1 kg of dried marigoldflower petals. The solvent is removed and the residue is dissolved in ahot alcohol. The solution is then filtered and then the lutein fattyacid ester is precipitated out. To obtain a more digestible form oflutein from extracted marigold flower petals, the extract is saponifiedat high pH (10+) or hydrolyzed to convert the product to a free formlutein.

U.S. Pat. No. 5,382,714, reports using commercially available saponifiedmarigold oleoresins to crystallize lutein after saponification of theoleoresins using organic solvents. Purification of lutein fromsaponified marigold oleoresins without the use of added organic solventsis reported in U.S. Pat. No. 5,648,564.

There are several drawbacks to the extraction methods reported above.For example, the method reported in U.S. Pat. No. 5,648,564 usescaustic, high pH conditions that may be dangerous and may cause yieldlosses and vapor exposure, as well as producing toxic waste materialsthat need to be disposed of when completed. Trace amounts of these toxicchemicals and solvents may be present in the final products, which maybe a problem for use of the resulting lutein extract for humanconsumption. The method reported in U.S. Pat. No. 5,382,711 uses organicand caustic solvents such as hexane, propane diol, and potassiumhydroxide for extraction and saponification processes, which may not betotally removed during the purification process. Furthermore, neithermethod utilizes a starting material in which lutein is obtained in itsfree form. As previously noted, free form carotenoids such as lutein mayprovide better adsorption into the body during consumption. Thus, itwould be desirable to provide a lutein extraction method that isolatesthe free form lutein without requiring the use of organic solventsduring any steps, from the extraction of lutein from raw materials tothe production of free lutein for consumption.

Lutein is abundantly present in a free, non-esterified form in greenplants such as alfalfa, broccoli, green beans, green peas, lima beans,cabbage, kale, spinach, collards, mustard greens, turnip greens, kiwi,and honeydew. Green plants may also be rich in a variety of additionalnutrients. For example, alfalfa is rich in proteins, minerals, andvitamins. It contains all 21 amino acids, and is very high in vitaminsA, D, E, B-6, and K, calcium, magnesium, chlorophyll, the fatty acidslinolenic and linoleic, phytoestrogens, phosphorous, iron, potassium,trace minerals and several digestive enzymes. It also contains severalsaponins, many sterols, flavonoids, coumarins, alkaloids, acids,additional vitamins, amino acids, natural sugars, proteins (25% byweight), minerals, trace elements and other essential nutrients.

Extraction of lutein from green plants may be beneficial because itremoves the need for the additional chemical step of saponification orester cleavage to release the free lutein, which is the desired form forbest absorption as consumed. However, the isolation and purification oflutein from plants has not been economical in the past because manyexpensive and time-consuming purification steps have been required toseparate the lutein from the large quantities of other compounds presentin the plant materials. (U.S. Pat. No. 5,382,714).

Supercritical fluids (SCF), which are gases above their criticalpressure and temperature, have been used in certain industries toperform extractions. SCFs are dense gasses in a separate phase, which isdistinct from normal gas phase. SCFs have a density and solvating powersimilar to that of a liquid and diffusion rates similar to that of agas. Supercritical fluids are unlike liquids because their solvent poweris highly sensitive to pressure changes and may be varied over widelimits by changing the pressure.

SCF extraction offers a relatively rapid, simple and inexpensivetechnique to perform purification or compound preparations. Mostcompounds, once dissolved, can quickly and cleanly be precipitated orremoved from the supercritical fluids by lowering the pressure and/ortemperature or both to achieve separation. Because a slight change inthe pressure or temperature of a system causes significant change insolubility, the use of SCF enables a highly efficient isolationprocedure of the desired components to be extracted. Using the method ofpostextraction fractionation with a column designed to allow fortemperature and pressure drops at different levels to gain the desiredresults may effect further concentration and purification.

One method of extracting carotenoids such as lutein from alfalfa withoutusing toxic solvents is reported in Favati et al., Supercritical CO₂Extraction of Carotene and Lutein from Leaf Protein Concentrates (1988).In the method reported in Favati, extracts containing mixtures of freelutein and β-carotene were obtained from alfalfa by supercriticalextraction in a single stage extractor. This laboratory scale extractionwas done in a single step, extracting a mixture of lutein, carotene, andother components from a leaf protein concentrate, with the relativeconcentrations of the two carotenoids dependent upon the extractionpressure used. The carotenoid content obtained from the process was 1.5%of the total extract.

Although the supercritical extraction method reported in Favati et al.overcomes the aforementioned problems with the health and safety risksof conventional solvent extractions, the resulting extracts include amixture of lutein and other carotenoids in a single extraction. However,given the beneficial health effects of lutein, it would be desirable toobtain an isolated lutein extract containing a substantial concentrationof lutein while being substantially free of other carotenoids.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method for isolatinglutein from green plant materials, in which a first supercritical fluidextraction of the green plant material is performed at a first pressureto obtain a first extract. A second supercritical fluid extraction ofthe green plant material is then performed to obtain a second extract.The second extract includes lutein, but is substantially free ofcarotenes such as β-carotene. The second extract is then separated fromthe supercritcal fluid used to perform the first and secondsupercritical extractions. The first extract may be separated from thesupercritical fluid in a similar manner.

A variety of green plant materials may be used as the starting materialin the method of the present invention. Suitable green plant materialsmay include alfalfa, wheat grass, barley grass, broccoli, kale, spinach,cabbage, soybeans, green beans, mustard greens, turnip greens, collards,and green peas. In one embodiment, alfalfa is provided as the greenplant material.

During the first and second supercritical extractions of the green plantmaterial, the first and second pressures may be between about 8 MPa toabout 200 MPa, more particularly between about 10 MPa to about 120 MPa.In one embodiment, the first pressure is lower than the second pressure.For example, the first pressure may be between about 10 and about 40MPa, and the second pressure may be between about 41 and about 80 MPa.More particularly, the first pressure may be about 20 Mpa and the secondpressure may be about 65 Mpa. The temperature during the supercriticalextractions may be between about 31° C. to about 200° C., moreparticularly between about 31° C. to about 40° C., or even moreparticularly about 35° C. The temperature may be varied or remainconstant during the extractions

By optimizing the temperature and pressure at which the first and secondsupercritical extractions are performed, each extract may contain asubstantial concentration of a particular substance, such as a desiredcarotenoid. In one embodiment, the first extract includes a substantialamount of β-carotene and the second extract includes a substantialamount of lutein, but is substantially free of β-carotene. In anotherembodiment, the first supercritical extraction is performed until thegreen plant material is substantially free of β-carotene. Additionalextractions may also be performed at additional pressures and/ortemperatures.

After performing the second supercritical extraction, the second extractmay be separated from the supercritical fluid by lowering the pressureof the second extraction such that the lutein precipitates out of thesecond extract and onto a desired carrier. The first extraction may beseparated in a similar manner. In one embodiment, the pressure of thefirst extract may be lowered to about 10 MPa and the pressure of thesecond extract may be lowered to about 40 MPa. The first or secondextract may then be processed to form an end product suitable forconsumption.

In another embodiment, the present invention provides a continuousmethod for obtaining a plurality of extracts from a green plantmaterial. A plurality of supercritical extractions may be performed at aplurality of pressures to obtain a plurality of extracts. For example,one of the extracts may contain substantial amounts of lutein. Anotherextract may contain substantial amounts of carotene. Other extractionsmay contain fatty acids, xanthophylls, zeaxanthin, astaxanthin,canthaxanthin, capsorubin and cryptoxanthin. Such extractions may beobtained by optimizing the pressure and/or temperature environment atwhich the extract is obtained to provide an extract having a substantialconcentration of the desired substance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a flow-chart for the fractionation and extraction oflutein according to an embodiment of the present invention.

FIG. 2 illustrates a lutein extraction chamber and a process flowdiagram with collection system according to an embodiment of the presentinvention.

DETAILED DESCRIPTION

The present invention provides a method of extracting carotenoids suchas lutein from green plant materials using a supercritical extractionprocess. The process is optimized as to the pressure and/or temperatureduring extraction to obtain the highest concentration of the desiredcarotenoids.

The green plant material may be derived from any suitable green plantmaterial, including alfalfa, wheat grass, barley grass, broccoli, kale,spinach, cabbage, soybeans, green beans, mustard greens, turnip greens,collards, or green peas. A wet or dried chloroplast rich fraction of agreen plant may be particularly useful for the extraction ofcarotenoids. The chloroplast rich fraction may be separated from otherplant fractions by a process that includes the use of heat, acids,centrifugation, electrical field, or flocculants. The chloroplast richfraction may be dried to 5-50% moisture with hot air, infrared heat,microwave radiation or a vacuum oven prior to the extraction with supercritical fluid to preserve the desired components. Prior to extraction,the chloroplast rich fraction may be washed with an aqueous solutionprior to the extraction with super critical fluid. The washing step mayremove bitter flavors from the chloroplast rich fraction to provide amore palatable fraction for use in a nutritional supplement.

Pre-Extraction Processing of Green Plant Material

The green plant materials may be processed in a variety of ways prior toperforming supercritical extraction to obtain a desired startingmaterial. In one embodiment, the green plant material is subjected tothe wet fractionation process illustrated in FIG. 1. In this embodiment,pre-bloom alfalfa may be harvested with standard farm equipment and thencut or chopped into ½ to 4-inch lengths. This cutting or choppingprocess is generally performed within 1 hour after harvesting topreserve the desired compounds. The cut or chopped alfalfa may then becrushed or macerated with rollers or with hammermill devices thatspecifically ruptures the plant cell walls. The macerated green crop maythen be squeezed in an appropriate pressing device, screw press, orother press that separates the green plant juices from the fibrous plantmaterial.

The residue fibrous plant fraction, or wet fiber fraction of alfalfatypically possesses 55-65% moisture, 14-18% protein, and has most of thetypical nutritional value of green forages. This fraction may be usedfor ruminant feed for beef or dairy cows in either wet or dry form.

The green plant juice is a mixture of cell sap materials, which includewater, salts, chloroplasts, and cytoplasmic proteins, enzymes and cellcompounds. The juice may be further treated by one of several methods toseparate desired components. In one embodiment, the juice is typicallysubjected to heat coagulation at 60° C. for the chloroplast fraction andat 85° C. for the cytoplasmic fraction. Alternatively, the juice may betreated by acid precipitation, by density separations in centrifugalfields, or by direct electrical current fields. These techniques producethree general fractions: (a) a green protein chloroplast fraction; (b) awhite cytoplasmic protein fraction; and (c) a brown juice fraction. Inone embodiment, separation of the green protein concentrates from thebrown juice is performed by centrifugation or filtration methods.

In one embodiment, the green protein chloroplast fraction of alfalfa isthe starting green plant material for the supercritical extraction oflutein from green plants using supercritical fluid. This fraction isrich in plant chloroplasts and is typically composed of 50-55% proteinon a dry weight basis and has 1.2 to 2.8 g xanthophylls per kg. Thegreen chloroplast fraction may be used wet or may be dried prior toextraction of carotenoids. The dried form may produce a more stablematerial for extraction.

The fractions of the green plant juice may be dried under gentleconditions to preserve the desired components. Drying may beaccomplished with hot air or other hot inert gases, infrared heat,microwave, vacuum oven devices, or any other method or combination ofthese to remove water to the desired level.

Washing the green protein chloroplast fraction with an aqueous solutionor water just prior to supercritical extraction may be advantageous.This washing process may remove off-flavors and bitter grassy flavorsfrom the protein concentrate fraction and may make the extract morepalatable for subsequent human consumption. Lutein has very littlesolubility in water, so the water wash causes only minor loss ofproduct. This washing step may be particularly beneficial if thepost-extraction green protein chloroplast fraction is used as part of anutritional supplement.

Although alfalfa is used as the green plant material in the reportedembodiment, any fresh green crop that can be processed by wetfractionation may be used, including wheat grass, barley grass,broccoli, kale, spinach, cabbage, soybeans, green beans, mustard greens,turnip greens, collards, or green peas. For example, the wetfractionation process reported above may be easily adapted to wheatgrass and barley grass. Since the wet fractionation is similar foralfalfa and grasses the process is the same for most fresh green plants.

Supercritical Extraction Process

Once a suitable green plant material is obtained, supercriticalextraction may be performed by passing supercritical fluids (SCF)through the green plant material. The supercritical fluid used in themethod of the present invention may include CO₂, CH₂CH₂, CH₃CH₃, N₂O orother suitable supercritical fluids. A co-solvent may be used along withthe supercritical fluid to increase the solvation power for polaranalytes that do not readily dissolve in supercritical fluids.Co-solvents are often referred to as entrainers or modifiers, and aretypically a liquid organic solvent such as methanol, ethanol, propylenecarbonate, acetone, tetrahydrofuran, formic acid, propylene glycol, orethyl acetate that are blended with the carbon dioxide. With anentrainer, the solvent system has a much higher polarity and is able tosolubilize more polar analytes for extraction. Entrainers have beenshown to substantially increase the solubility of zeaxanthin insupercritical carbon dioxide (U.S. Pat. No. 5,747,544). In oneembodiment, the SCF includes ethanol as an entrainer at 1-5%concentration in the extracted material. This entrainer may produce abetter extraction at lower pressures.

In one embodiment, the SCF is carbon dioxide, which has a criticalpressure of 1070 psi (about 7.4 MPa) and a critical temperature of 31°C. Solvation power increases as pressure and temperature is raised abovethe critical pressure and temperature. Supercritical CO₂ may bemanipulated at room temperature, making the handling of heat-vulnerablesubstances easy and safe. Fire and explosion hazards associated withlarge-scale extractions using organic solvents are eliminated with thissolvent.

In practice, the fluid is passed through the green plant materialsinside an extraction vessel. The fluid diffuses into the pores of thegreen plant material matrix, solubilizes the extracts (e.g., lutein orcarotene) of interest, and then carries the extracts away from the greenplant matrix in a solution. The extract is then collected, and the greenplant matrix (now without the extract) is left behind in the extractionvessel. Supercritical fluids have favorable diffusion and viscositycoefficients providing for good mass transfer characteristics. Changingfluid pressure or temperature may control solvent strength in aprecisely controlled manner. As opposed to conventional solventextraction, any residual CO₂ left in the extract after separation isinert and non-toxic, such that human consumption of the material is notharmful.

The extraction process illustrated in FIG. 2 is typically performed in around thick-walled very high-pressure chamber, engineered to withstandpressures up to about 120 MPa (1450-17,400 psi), more particularly up toabout 70 MPa ( 10,150 psi). The chamber has openings for adding asuitable charge of green plant protein concentrate at the top and forremoval of the charge after extraction at the bottom. Appropriate pipesand pump systems direct the supercritical carbon dioxide fluid into thebottom of the chamber such that the liquid will flow up through the bedof green plant material and to the top of the chamber for delivery to acollection device. During or after delivery of the extract to thecollection device, the supercritical fluid may be depressurized to belowthe desired pressure to collect the desired extract. In one embodiment,the extraction method is performed by counterflowing the SCF relative tothe movement of the green plant material.

Importantly, the temperature and pressure are controlled with theconventional devices or heat exchangers before, during and/or afterextraction to optimize the isolation of lutein or other extracts. Afterleaving the extraction chamber, a pressure reduction valve may bepositioned prior to the collection device intake to effect release orprecipitation of the desired extract alone, or onto a specific carriermaterial in the collection device. A double valve at the bottom of thecollection device allows for periodic removal of the extract (with orwithout the carrier). The vented carbon dioxide liquid from the top ofthe collection device at a reduced pressure may then be recycled to afilter system and recompressed to high pressure for use in a secondextraction function in the extraction vessel. Extraction is continueduntil an appropriate degree of desired product is isolated from theplant material being processed. The volume of SCF needed for the desiredextraction depends on the pressure and temperature used for each productobtained. Typically 5-8 cubic feet of SCF are needed for each cubic footof plant concentrate extracted.

In one embodiment, the supercritical extraction is performed at leasttwo different pressures within the extraction chamber. At a firstpressure, a first extract containing substantial amounts of carotene maybe obtained. At a second pressure, a second extract containingsubstantial amounts of lutein is obtained. Advantageously, the secondextract may be substantially free of β-carotene. For example, the secondextract may have less than 10 percent lutein, more particularly lessthan 5 percent lutein. This may be accomplished by performing the firstextraction until the green plant material is substantially free ofβ-carotene, and then subsequently performing the second extraction untilthe desired lutein extraction is completed.

Post-Extraction Processing

Optionally, after separation, the extract may be further processed toproduce a desired end product. For example, a secondary columnfractionation step may be used to further concentrate and purify luteinor β-carotene. Additionally, the first or second extract may be purifiedwith simple non-toxic solvents such as food grade ethanol, a vegetableoil, or water to provide a substance that is crystalline and essentiallypure and free of any potentially toxic chemicals, even on a trace level.Typically, the lutein is concentrated to 5-50% concentration in oils ordry form for bulk markets. In one embodiment, the first and secondextracts are combined before or after separation in order to provide anend product having a controlled concentration of carotene and lutein.

The lutein or β-carotene may be also further processed by blending ormilling with a suitable base material (e.g. green plant proteinconcentrate) to form an end product suitable for human consumption. Thisblending or milling step may take place in the collection device whereinthe extract is precipitated into the base material. The double valve atthe bottom of the collection device may then be actuated to release theblended extract.

End Products

The extract may be combined with a suitable carrier to form an endproduct. The end product may be a powder, an agglomerated powder or asolution in edible oil that includes the extract. A protein matrix orbeadlets may be produced to protect the extract from deterioration oroxidation. It may be analyzed for specific carotenoid content and thenmixed with alfalfa or plant based natural fillers, sugars, gelatins, orstarches to form a desired standardized dry product. In one embodiment,the extract is combined with a green chloroplast rich fraction ofalfalfa (which may also be used as the starting green plant material)such that an end product will contain only a single source ingredientand may be labeled as 100% alfalfa based. The use of the greenchloroplast fraction of alfalfa as the carrier in the final product isnutritionally beneficial because of the high content of useful proteins,vitamins, amino acids, chlorophyll and other compounds in the fractionin addition to the presence of the concentrated lutein. Furthermore, theend product is then derived from a single source plant product, withoutadditional fillers or additives.

The invention is further described in the Example below.

EXAMPLE

Fresh field chopped alfalfa was run through a hammermill to ruptureplant cells. The tip speed of the hammers was set at 15,000 feet perminute to crush the green wet (80% moisture) material without causingthe material to be pulped or broken into smaller pieces. The crushedmaterial was run through a single (6″) screw press (Model Number VP6,available from Vincent Corp., Tampa, Fla.) such that the outletrestriction (set at 25 psi) produced high continuous pressure to effectseparation of green plant juices from the plant fibers. The long barrelscrew has a fine barrel screen to allow juice to flow from the fiber.The ratio of juice to fiber was about 1:1, however, the yield of juiceto fiber will be less if the starting material is old or more matured,or if it is naturally dryer than lush pre-bloom growing alfalfa. Thejuice was adjusted to a pH of 8.0 with ammonia water, and immediatelyheated from ambient temperature with a double boiler system with apropane burner such that the juice was heated within 5-10 minutes afterproduction to between 82-85° C. to cause heat coagulation of the greenand white (cytoplasmic) proteins. The green protein coagulum wasseparated with a weir type screen to separate the green “curd” from thebrown waste plant juices.

The green wet protein “curd” (i.e. the green plant material) wasimmediately dried in a continuous perforated temperature controlled zonedryer such that limited heat (below 85° C.) with limited air at 5-10%relative humity produced a dry granular material. The wet protein curdstarted at 65% moisture and was dried to 8% moisture. This material wasthen extracted or stored in oxygen-excluding bags or containers in thedark at room temperature until extracted.

The green plant material was then transferred to a very high pressureextraction chamber (about 5 cm×50 cm) having round thick-walls, andbeing engineered to withstand pressures of up to about 70 MPa (10,150psi). The chamber was brought up to pressure and temperature with 20 MPacarbon dioxide fluid at 30° C. The temperature and pressure of the SCFstream with an injected 3% liquid ethanol (vol./vol.) entrainer wasregulated by a high pressure carbon dioxide pump and heat exchangercontrolled with water in a tube and shell system. This extractioncontinued until the beta-carotene (1-4-bed volumes) was removed asmeasured in side port sampling at the top of the column outlet line. Thepressure was then increased to 65 MPa to extract the lutein from thegreen plant material with 10 bed volumes.

The desired compounds were collected after extraction into a small buttall (1 meter) tower with reduced pressure through reducing valves suchthat the beta-carotene and lutein fractions were collected into chamberswith dried green protein powder at 10 MPa and 40 MPa respectively. Thelower ¼ of the collection vessel has large valves to allow the desiredfractions to fall out into the protein fractions such that after theseparation of the fractions, the lower collection chamber was sealed offand the pressure released to remove the end products.

The yield in this example was 2.6 grams beta-carotene and 2.4 grams oflutein per kilogram of dry (6% moisture) starting material. The productswere tested for purity without the blending with the green proteinfraction with silica gel HPLC columns and were 80% and 72% pure caroteneand lutein respectively. The drying of the green protein is critical inpreserving the desired end products since the dried materials rangedfrom 0.6 to 3.4 grams of each carotenoid as measured with highperformance liquid chromatography (HPLC) with known pure standards(available from Sigma Chemical, St. Louis, Mo.).

What is claimed is:
 1. A method for isolating lutein from green plantmaterials comprising: performing a first supercritical fluid extractionof a green plant material at a first pressure to obtain a first extract;performing a second supercritical fluid extraction of the green plantmaterial at a second pressure to obtain a second extract that includeslutein, but is substantially free of carotene; and separating the secondextract from the supercritical fluid.
 2. The method of claim 1 whereinthe green plant material is derived from alfalfa, wheat grass, barleygrass, broccoli, kale, spinach, cabbage, green beans, soybean plants,mustard greens, collards, or turnip greens.
 3. The method of claim 1,wherein the green plant material comprises a wet or dried chloroplastrich fraction of alfalfa.
 4. The method of claim 3, wherein thechloroplast rich fraction is formed by heating green plant liquid tocause coagulation of a chloroplast rich fraction and a cytoplasmic plantfractions and separating the chloroplast rich fraction from brown plantjuices.
 5. The method of claim 3, wherein the chloroplast rich fractionis dried with hot air, infrared heat, microwave radiation or a vacuumoven prior to performing supercritical fluid extraction.
 6. The methodof claim 3, the chloroplast rich fraction is washed with an aqueoussolution prior to performing supercritical fluid extraction.
 7. Themethod of claim 1 wherein the first extract comprises β-carotene.
 8. Themethod of claim 1 wherein the first supercritical fluid extraction isperformed until the green plant material is substantially free ofβ-carotene.
 9. The method of claim 1 wherein the first and secondpressures are between about 10 and about 120 MPa.
 10. The method ofclaim 1 wherein the first pressure is lower than the second pressure.11. The method of claim 1 wherein the first pressure is between about 10and about 40 MPa.
 12. The method of claim 1 wherein the second pressureis between about 41 and about 80 MPa.
 13. The method of claim 1 whereinthe first pressure is between about 15 and about 35 MPa and the secondpressure is between about 55 and about 80 MPa.
 14. The method of claim 1wherein the first and second supercritical fluid extractions occur atbetween about 30° C. and about 100° C.
 15. The method of claim 1 whereinthe first and second supercritical fluid extractions occur at about 35°C.
 16. The method of claim 1 wherein the first and second supercriticalfluid extractions occur at a substantially constant temperature.
 17. Themethod of claim 1, wherein the first and second supercritical fluidextractions are performed with a supercritical fluid comprising CO₂,CH₂CH₂, CH₃CH₃, N₂O or mixtures thereof.
 18. The method of claim 1,wherein the first and second supercritical fluid extractions areperformed with a supercritical fluid and an entrainer.
 19. The method ofclaim 18, wherein the entrainer comprises ethanol.
 20. The method ofclaim 18 wherein the entrainer comprises a concentration of betweenabout 1 to 5 (vol/vol) percent of the supercritical fluid.
 21. Themethod of claim 1 wherein separating the second extract from thesupercritical fluid comprises subjecting the second extract to apost-extraction pressure that is lower than the second pressure.
 22. Themethod of claim 21 wherein the post-extraction pressure is between about35 and about 45 MPa.
 23. The method of claim 1 further comprisingseparating the first extract from the supercritical fluid.
 24. Themethod of claim 23 comprising separating the first extract from thesupercritical fluid by subjecting the first extract to a post-extractionpressure that is lower than the first pressure.
 25. The method of claim1 further comprising processing the first or second extract by columnfractionation.
 26. The method of claim 1 further comprising processingthe first or second extract by purifyng with a non-toxic solvent. 27.The method of claim 1 further comprising processing the first or secondextract by blending with dried green protein.
 28. The method of claim 1comprising combining the first and second extract.
 29. The method ofclaim 1, further comprising performing a low pressure extraction priorto performing the supercritical fluid extractions.
 30. The method ofclaim 1 further comprising recycling the supercritical fluid.
 31. Themethod of claim 1 comprising performing at least a third supercriticalfluid extraction of the green plant material to obtain at least a thirdextract, wherein each supercritical fluid extraction is performed at adifferent pressure.
 32. The method of claim 31 wherein the pressure usedin the third supercritical fluid extraction is lower than the first andsecond pressures.
 33. The method of claim 31 wherein the pressure usedin the third supercritical fluid extraction is lower than the secondpressure but higher than the first pressure.
 34. A method for obtaininga plurality of extracts from a green plant material comprising providingthe green plant material; performing a plurality of supercritical fluidextractions of the green plant material at a plurality of differentpressures to form a plurality of extracts, wherein one of the pluralityof extracts includes lutein and is substantially free of carotene; andseparating at least one of the plurality of extracts from thesupercritical fluid.
 35. The method of claim 34 wherein at least one ofthe plurality of extracts comprises β-carotene.
 36. The method of claim34 wherein at least one of the plurality of extracts comprisesalphacarotene.
 37. The method of claim 34 wherein at least one ofplurality of extracts comprises free fatty acids.
 38. The method ofclaim 37 wherein the fatty acids comprise a linolenic acid, linoleicacid palmitic acid or oleic acid.
 39. The method of claim 34, wherein atleast one of the plurality of extracts comprises xanthophyll.
 40. Themethod of claim 34, wherein at least one of the plurality of extractscomprises zeaxanthin.
 41. The method of claim 34, wherein at least oneof the plurality of extracts comprises astaxanthin.
 42. The method ofclaim 34, wherein at least one of the plurality of extracts comprisescanthaxanthin.
 43. The method of claim 34, wherein at least one of theplurality of extracts comprises capsorubin.
 44. The method of claim 34,wherein at least one of the plurality of extracts comprisescryptoxanthin.